This invention relates to nucleic acid and amino acid sequences of a new mammalian protein and to the use of these sequences in the characterization, diagnosis, prevention, and treatment of cell proliferative disorders.
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of biochemical and physiological mechanisms and metabolic pathways. Despite different evolutionary pressures, proteins that regulate the cell cycle in yeast, nematode, fly, rat, and man have common chemical or structural features and modulate the same general cellular activity. Comparisons of human gene sequences with those from other organisms where the structure and/or function may be known allow researchers to draw analogies and to develop model systems for testing hypotheses. These model systems are of great importance in developing and testing diagnostic and therapeutic agents for human conditions, diseases and disorders.
Signal transduction cascades alter gene expression by activation or suppression of transcription factor activity. Two basic types of transcription factors exist within the cell. Steroid hormone receptors are transcription factors whose activity is regulated by binding to lipid soluble hormones, such as steroids, retinoids, and thyroid hormones. Nuclear receptors are transcription factors, such as CREB (cAMP-response element binding protein), STAT (Signal Transducers and Activators of Transcription), and the TCF (ternary complex factor)-SRF (serum-response factor) complex, whose activity is regulated by phosphorylation cycles. The kinases and phosphatases that regulate the phosphorylation cycle respond to extracellular signals, such as growth factors and cytokines.
Hormone binding or phosphorylation induce conformational changes in transcription factors which promote their association with a diverse group of nuclear transcription factor binding proteins (TFBPs) including steroid receptor co-activator (SRC)-1, transcriptional intermediary factor (TIF), and CREB binding protein (CBP)/p300. These TFBPs function as modulators of transcription and show specificity towards transcription factor and associated ligands. For example, PIAS (protein inhibitor of activated STAT)-3 preferentially binds phosphorylated Stat3. Furthermore, IL-6 but not interferon-xcex3 stimulates interaction between Stat3 and PIAS3 (Chung et al. (1997) Science 278:1803-1805). Many TFBPs also have a restricted tissue distribution. For example, xcex2-3 adrenergic agonists activate peroxisome proliferator-activated receptor (PPAR)-xcex3. PPARxcex3 is a major regulator of fat cell-specific gene regulation and differentiation, but does not inherently distinguish whether fat cells proceed along energy storage (white fat) or energy dissipation (brown fat) pathways. PGC- 1, a modulator of PPARxcex3, is found only in brown fat and leads to specific activation of genes associated with energy dissipating adaptive thermogenesis (Puigserver et al. (1998) Cell 92:829-839).
A short sequence motif LXXLL is necessary and sufficient to mediate binding of TFBPs to activated nuclear receptors (Heery et al. (1997) Nature 387:733-736). The motif forms an xcex1-helix and occurs at the boundary of nuclear receptor interaction domains. ARIP3, which contains two LXXLL motifs, is an androgen receptor binding protein expressed predominantly in the testis which provides tissue specific gene activation by androgens (Moilanen et al. (1999) J. Biol. Chem. 274:3700-3704). ARIP3 is a member of a family of related TFBPs which include the PIAS proteins and Gu/RNA helicase II-binding protein (GBP). These proteins show 60-80% homology over the N-terminal and central regions of the proteins, but contain divergent C-termini. All of the PIAS family members also contain a potential zinc finger motif which is involved in transcription factor binding.
TFBPs, by binding and modulating transcription factor activity in a signal and tissue specific manner, provide additional regulation to cell signaling events. Inappropriate expression or activation of TFBPs can alter gene expression patterns and cell fates. AIB (amplified in breast cancer)-1, a member of the SRC1 family, interacts with the estrogen receptor and enhances estrogen receptor-dependent gene transcription. It is ubiquitously expressed in normal human tissues and is amplified and overexpressed in many breast and ovarian cancer cell lines and in breast cancer tumor samples (Anzick et al. (1997) Science 277:965-968). Mice lacking brown fat develop severe obesity and insulin resistance. Chronic exposure to xcex2-3 adrenergic agonists cause brown fat hypertrophy and thermogenic, anti-obesity effects due to enhanced activity of PGC 1 -PPARxcex3 (Puigserver, supra).
The discovery of a polynucleotide encoding a new mammalian protein satisfies a need in the art by providing new compositions which are useful in the characterization, diagnosis, prevention, and treatment of cell proliferative disorders.
The invention is based on the discovery of a polynucleotide encoding a mammalian protein (TFRP) which satisfies a need in the art by providing new compositions useful in the characterization, diagnosis, prevention, and treatment of cell proliferative disorders.
The invention provides an isolated and purified mammalian polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1 or a fragment thereof. The invention also provides fragments homologous to the mammalian polynucleotide from rat, mouse, and monkey.
The invention further provides an isolated and purified polynucleotide or a fragment thereof which hybridizes under high stringency to the polynucleotide encoding the polypeptide. The invention also provides an isolated and purified polynucleotide or a fragment thereof having a nucleic acid sequence which is complementary to the polynucleotide encoding the polypeptide.
The invention further provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence to at least one of the nucleic acids of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample. In one aspect, the method further comprises amplifying the polynucleotide prior to hybridization. The polynucleotide or fragment thereof may comprise an element or target on a microarray. The invention also provides a method for screening a plurality of molecules for specific binding to a polynucleotide or a fragment thereof, the method comprising providing a plurality of molecules, combining the polynucleotide of claim 1 with a plurality of molecules under conditions suitable to allow specific binding, and detecting binding of the polynucleotide to each of a plurality of molecules, thereby identifying at least one molecule which specifically binds the polynucleotide. Such molecules are potential regulators of polynucleotide function.
The invention also provides an expression vector containing at least a fragment of the polynucleotide of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell. The invention further provides a method for producing a polypeptide, the method comprising the steps of culturing the host cell under conditions suitable for the expression of the polypeptide and recovering the polypeptide from the host cell culture. The invention also provides an isolated and purified polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a portion thereof. Additionally, the invention provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:2 or a portion thereof in conjunction with a suitable pharmaceutical carrier.
The invention further provides a method for using a portion of the polypeptide to produce antibodies. The invention also provides a method for using a polypeptide or a portion thereof to screen for molecules which specifically bind the polypeptide, the method comprising the steps of combining the polypeptide or a portion thereof with a plurality of molecules under conditions suitable to allow complex formation and detecting complex formation, wherein the presence of the complex identifies a molecule which specifically binds the polypeptide. In one aspect, a molecule identified using the method increases the activity of the polypeptide. In another aspect, a molecule identified using the method decreases the activity of the polypeptide.